The objectives of this grant proposal are to study the clinical significance of pro-insulin, pro-insulin intermediates and C-peptide and to expand investigations of families with structurally abnormal proinsulin and insulin molecules. Specific antisera will be raised to human proinsulin in guinea pigs and rabbits and characterized in terms of their cross reactivity with insulin, C-peptide, proinsulin and its intermediates. Monoclonal antibodies will be raised and characterized similarly. These immunoassays will be used in conjunction with high pressure liquid chromatography (HPLC) to separate and identify serum proinsulin, its intermediates, C-peptide and insulin. In addition urinary C-peptide and its potential degradation products in dogs and man will be separated by a similar combination of techniques. The availability of human proinsulin and C-peptide will permit studies on their biological activity and metabolism. The potential use of human proinsulin for therapeutic purposes in Type I diabetics will be assessed. In these experiments, the possible in-vivo conversion of proinsulin to its intermediates will be evaluated using HPLC. The C-peptide will be used to study its metabolic clearance rate in man, as well as its urinary excretion. These studies should provide data relevant to accurately measuring insulin secretion in man. Several patients with mutant (pro)insulins have been identified. These patients and their families will undergo clinical evaluation in terms of their carbohydrate homeostasis. In addition their serum (pro)insulins will be characterized and the structural abnormality identified at both the protein and gene level. Studies in dogs will be carried out to quantify the metabolism of the abnormal insulin mutants and relate the results to the findings in the affected patients and their families.